Phosphoenolpyruvate carboxykinase (PEPCK) was discovered in 1953 by Utter and Kurahashi, yet more than 50 years later its metabolic role is still being resolved. PEPCK is a cataplerotic enzyme, which catalyzes the key reaction in hepatic and renal gluconeogenesis;however, it is present in mammalian tissues that do not make glucose. PEPCK-C is involved in a pathway termed glyceroneogenesis, a component of the triglyceride/fatty acid cycle. This cycle is a major factor in determining the levels of free fatty acids (FFA) in the blood and thus plays a role the genesis of insulin resistance and Type 2 diabetes in humans. During fasting, obese humans have three to four times the level of circulating FFA. Approximately 60% of the FFA that is released by lipolysis is re-esterified to triglyceride as part of the cycle;this is especially important in the liver, which has a high rate of FFA re-esterification. The major source of the 3- phosphoglycerol used for triglyceride synthesis is not glucose (via glycolysis), but pyruvate (via glyceroneogenesis). The pace-setting enzyme in glyceroneogenesis is PEPCK-C, which also occurs in muscle where it supports the turnover of triglyceride. As predicted from its role in glyceroneogenesis, the over-expression of PEPCK-C in muscle (PEPCK-Cmus mice) results in an accumulation of triglyceride at a level that is proportional to the activity of PEPCK-C in the muscle (the triglyceride can exceed 10 times the concentration in control muscle). PEPCK-Cmus mice eat twice as much as control littermates, but weigh 20% less and have more mitochondria. They have an extraordinary capacity for strenuous exercise;untrained PEPCK-Cmus mice can run continuously for up to 6 k, at a treadmill speed of 20m/min (untrained controls stop at 0.5 m), using the stored triglyceride as an energy source. Despite the dramatic effect of PEPCK-C on muscle metabolism and exercise capacity, there is virtually no information about the factors that control the levels of PEPCK-C in specific muscle types. The research proposed in this grant application aims to resolve many of the issues concerning the role of PEPCK in mammals, with the broad goal of relating its function with the control of triglyceride/fatty acid cycling in humans.